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. 2016 Dec 21;12(12):e1006089. doi: 10.1371/journal.ppat.1006089

Fig 12. Silencing of Ehnpc1 and Ehnpc2 genes in E. histolytica.

Fig 12

(A) Trophozoites clone G3 were transfected with psAP-2, psAP-2Ehnpc1 or psAP-2Ehnpc2a plasmids and stable populations were selected with 4 μg/ml G-418. RT-qPCR assays were performed using mRNA from transfected trophozoites (empty vector, KD Ehnpc1 and KD Ehnpc2), using specific primers for Ehnpc1 and Ehnpc2a genes and as a housekeeping the 40s ribosomal S2 protein gene. (B) Western blot assays of transfected trophozoites extracts, using α-EhNPC1 and α-EhNPC2 antibodies and respectively secondary antibody. As a loading control, the same membrane was reblotted with α-actin antibodies. (C) Densitometry analysis of bands showed in (B) normalized again actin protein. (D) Confocal microscopy of transfected trophozoites in basal conditions using α-EhNPC1 or α-EhNPC2 antibodies. Ph c: phase contrast. (E) Cholesterol concentration in the transfected trophozoites was measured as described in material and methods. (F) Rate of erythrophagocytosis of transfected trophozoites. * p<0.05, ** p<0.01. (G) Diaminobenzidine-stained trophozoites that ingested erythrocytes for different times. (H) Motility assays of transfected trophozoites cultured in transwell inserts. *** p<0.001.